A DNA-Binding Peroxiredoxin of Coxiella burnetii Is Involved in Countering Oxidative Stress during Exponential-Phase Growth

Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen''s proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100°C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain''s ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen''s early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella''s metabolism during intracellular replication.Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. Growth of the pathogen is restricted to an intracellular lysosome-like compartment, termed a parasitophorous vacuole (PV). The pH (∼4.5) of the PV is ideal for C. burnetii''s acidophilic lifestyle (11, 12); however, the effect of concomitant oxidative stress on the bacterium and the defense mechanisms used to counter it have not been well characterized.Bacteria deal with oxidative stress by employing antioxidants such as glutathione, vitamins A, C, and E, and carotenoids, and they counter reactive oxygen species (ROS) by using a variety of enzymatic effectors, including superoxide dismutase (SOD), catalase, and peroxidase. Previous work suggests that catalase and SOD are potential persistence factors for Coxiella bacteria residing in the intracellular niche (1, 14). In addition, a secreted acid phosphatase possibly reduces the respiratory burst of the host cell by inhibiting NADPH oxidase (4, 23). in silico analysis of the C. burnetii RSA 493 genome (22) revealed predicted Mn- and Cu/Zn-SODs, catalase, and four peroxiredoxins (Prxs), which are antioxidant enzymes (EC 1.11.1.15) that are thiol-containing reductants used to detoxify hydroperoxides. Interestingly, certain C. burnetii strains possess a frame-shifted Cu/Zn-SOD gene (e.g., Dugway) or a markedly truncated catalase gene (Nine Mile or G) (18), suggesting that these strains, likely undergoing reductive evolution in the intracellular niche, must resort to alternative effectors for protection against ROS.For this report, we characterized the Coxiella BCP (CBU_0963), a representative of a 2-cysteine (2-Cys) Prx subfamily that is widely employed by pathogenic bacteria to deal with potentially toxic H₂O₂ and organic hydroperoxides. We demonstrate that the bcp gene of Coxiella bacteria is maximally expressed during early exponential-phase growth and that BCP is a DNA-binding protein that exhibits peroxidase activity and can protect supercoiled DNA from oxidative damage in vitro.